The PI3K/AKT signaling pathway, which is frequently dysregulated in cancer, controls key cellular processes such as survival, proliferation, metabolism, and growth. Additionally, protein glycosylation, the process by which carbohydrates are added to amino acids, is essential for proper protein folding and is also frequently deregulated in cancer. In cancer, cancer cells grow and proliferate at faster rates, and thus require increased protein folding capacity to sustain increased proliferation. The glycosyltransferase Asparagine-linked glycosylation 3 homolog (ALG3), which resides in the endoplasmic reticulum membrane, catalyzes the addition of mannose units to a glycan precursor once the glycan is flipped into the ER lumen during glycan biosynthesis. ALG3 function is a rate-limiting step during glycan biosynthesis and preliminary data from our laboratory show that it is a novel AKT substrate. Notably, we find that in both lung and breast cancer cells, ALG3 is phosphorylated downstream of PI3K and AKT. This represents, to our knowledge, the first identified link between PI3K/AKT oncogenic signaling and protein glycosylation in the context of cancer-specific signaling. Additionally, ALG3 resides proximal to the PIK3CA gene in the 3q26 amplicon. Consequently, PIK3CA and ALG3 are co-amplified in 89%, 28% and 76% of lung squamous cell carcinoma and breast and ovarian carcinoma, respectively. The hypothesis driving my proposed project is that ALG3 plays a key functional role in the regulation of protein N-glycosylation downstream of PI3K/AKT signaling, and that hyperactivated PI3K/AKT alters glycosylation, leading to functional consequences in cancer. Specifically, I postulate that cells that harbor PIK3CA amplification and ALG3 upregulation increase glycosylation and protein folding rates, allowing cells to cope with increased protein translation in response to hyperactive PI3K/AKT. The goal of this project is to determine the relationship between PI3K/AKT signaling and N-glycosylation. In Aim 1 I will assess the role of PI3K/AKT signaling in modulating glycosylation through ALG3 phosphorylation by using western blotting and signaling assays to investigate phosphorylation patterns, glycomics to investigate changes in the glycan profile, and peptido-glycomics and lectin staining assays to identify proteins affected by deregulated ALG3. In Aim 2 I will determine the functional consequences of deregulated ALG3 in PIK3CA- driven cancer using dependency mapping, proliferation assays, apoptosis assays, by measuring stress- response gene expression, and by using an in vivo model. This project will advance our understanding of the regulation of glycosylation by PI3K/AKT signaling and its role in cancer progression, and pave the way for exploring future combination strategies targeting PI3K/AKT and protein glycosylation.